Determining intracellular lipid content of different oleaginous yeasts by one simple and accurate Nile Red fluorescent method.

A simple and accurate Nile Red fluorescent method was built to evaluate the lipid content of three different oleaginous yeasts by one standard curve. The staining of cells can be observed clearly by laser scanning confocal microscope, showing that Nile Red can enter into the cells of oleaginous yeasts easily. A series of conditions such as pretreating temperature, cell suspension concentration (OD600), staining time, Nile Red concentration and the type of suspension solvent were learnt systematically to obtain the optimal process parameters for Nile Red staining. After optimization, the fitting curve of Nile Red fluorescent method was established under suitable conditions (pretreating temperature: 50 °C, OD600: 1.0; staining time: 5 mins; Nile Red concentration: 1.0 µg/mL; suspension solvent: PBS) and it had a suitable correlation coefficient (R2 = 0.95) for lipid content measurement of different oleaginous yeasts. By this study, the possibility of lipid content determination of different oleaginous yeasts by one fitting curve can be proven and this will improve the efficiency of researches related to microbial lipid production.

Neutralization of acidic drainage by Cryptococcus sp. T1 immobilized in alginate beads.

We isolated Cryptococcus sp. T1 from Lake Tazawa's acidic water in Japan. Cryptococcus sp. T1 neutralized an acidic casamino acid solution (pH 3.0) and released ammonia from the casamino acids to aid the neutralization. The neutralization volume was estimated to be approximately 0.4 mL/h. The casamino acids' amino acids decreased (1.24→0.15 mM); ammonia increased (0.22→0.99 mM). We neutralized acidic drainage water (1 L) from a Tamagawa River neutralization plant, which was run through the column with the T1-immobilized alginate beads at a flow rate of 0.5 mL/min, and observed that the viscosity, particle size and amounts of the alginate beads affected the acidic drainage neutralization with an increase of the pH value from 5.26 to 6.61 in the last fraction. An increase in the Al concentration decreased Cryptococcus sp. T1's neutralization ability. After 48 h, the pH of acidic water with 50 mg/L Al was apparently lower than that without Al. Almost no pH increase was observed at 75 mg/L.

MALDI-TOF MS for the identification of veterinary non-C. neoformans-C. gattii Cryptococcus spp. isolates from Italy.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an effective alternative to phenotypic and molecular methods for the rapid identification of microorganisms. Our aim in this study was to create an in-house library for a set of strains of nine uncommonly reported human and animal cryptococcal species, including Cryptococcus adeliensis, C. albidosimilis, C. albidus, C. aureus, C. carnescens, C. laurentii, C. magnus, C. victoriae and C. uniguttulatus, and to use this library to make timely and correct identifications using MALDI-TOF MS for use in routine laboratory diagnostics. Protein extracts obtained via the formic acid extraction method of 62 veterinary non-C. neoformans-C. gattii cryptococcal isolates were studied. The obtained mass spectra correctly grouped all 62 studied isolates according to species identification previously obtained by internal transcribe spacer sequence analysis. The in-house database was than exported and successfully uploaded to the Microflex LT (Maldi Biotyper; Bruker Daltonics) instrument at a different diagnostic laboratory in Italy. Scores >2.7 obtained from isolates reanalyzed in the latter laboratory supported the high reproducibility of the method. The possibility of creating and transferring an in-house library adds to the usefulness MALDI-TOF MS an important tool for the rapid and inexpensive identification of pathogenic and saprophytic fungi as required for differential diagnosis of human and animal mycoses.

Synthesis of magneto-sensitive iron-containing nanoparticles by yeasts.

Industrial production of magneto-sensitive nanoparticles, which can be used in the production of target drug delivery carriers, is a subject of interest for biotechnology and microbiology. Synthesis of these nanoparticles by microorganisms has been described only for bacterial species. At the same time, it is well known that yeasts can form various metal-containing nanoparticles used, for instance, in semiconductors, etc. This paper describes the first results of the biosynthesis of magneto-sensitive nanoparticles by yeasts. The organisms we used-Saccharomyces cerevisiae and Cryptococcus humicola-represented two different genera. Magneto-sensitive nanoparticles were synthesized at room temperature in bench-scale experiments. The study included transmission electron microscopy of the yeast cells and their energy dispersive spectrum analyses and revealed the presence of iron-containing nanoparticles. Both yeast cultures synthesized nanoparticles at high concentrations of dissolved iron. Electron microscopy showed that nanoparticles were associated mainly with the yeast cell wall. Formation of magneto-sensitive nanoparticles was studied under conditions of applied magnetic fields; a possible stimulating role of magnetic field is suggested. On the whole, the paper reports a novel approach to green biosynthesis of magneto-sensitive nanoparticles.

Comparison of torula yeast and various grape juice products as attractants for Mexican fruit fly (Diptera: Tephritidae).

Early research investigating attractants for the Mexican fruit fly, Anastrepha ludens Loew, during the 1930s indicated that fermentation products were effective attractants for Mexican fruit flies and other tropical Tephritidae, but that attraction to fruit components was only of academic interest. Tests reported here were carried out on populations of Mexican fruit flies from 2004 to 2011. Trapping experiments carried out at sites in the states Nuevo Leon and San Luis Potosi compared grape juice, reconstituted grape concentrate and powdered grape mixes, and torula yeast extract in orchards at each site. The Nuevo Leon orchard was mixed with alternate rows of pears and surrounded by alternate hosts. The San Luis Potosi site was surrounded by other orange orchards or nonhosts. Each test was run for at least 10 mo and included highest and lowest trapping periods. Results showed that grape juice captured the most total flies and had the fewest samples with zero flies. However, in the series of experiments, each product had the most captures in at least one experiment. Hydrolyzed torula was superior in one of the six experiments. In five of the tests, polyethylene glycol was tested as an additive to the grape products but never improved capture rate compared with the product without the additive. These results indicate that grape juice is superior to grape concentrate or powder and grape juice is at least equal to torula yeast hydrolysate for trapping pest populations of Mexican fruit flies in commercial citrus orchards.

Assessment of Cryptococcus albidus for biopulping of eucalyptus.

Cryptococcus albidus shows delignification activity in nature. It was used for the biopulping of eucalyptus wood (Eucalyptus grandis) to access its potential for industrial application in the pulp and paper industry. Enzyme analysis on days 15, 30, and 60 showed the presence of laccase and xylanase as key enzymes. The production of endo-glucanase (CMCase) and exo-glucanase (FPase) was very low. Scanning electron microscopy (SEM) showed the surface colonization of wood and loosening of wood fibers in C. albidus-treated samples. Fourier-transformation infrared spectroscopy (FT-IR) indicated the chemical modification of eucalyptus wood. Denaturing gradient gel electrophoresis (DGGE) analysis on days 15, 30, and 60 confirmed the presence of C. albidus throughout the experiments. Cryptococcu albidus was able to suppress the growth of a native population. Further, after 60 days both the control and treated eucalyptus wood chips were given kraft pulping treatment. The kappa number of pulp of control wood was 21 and for treated wood was 17. Kappa number is considered a measure of lignin content in wood; hence the treatment of eucalyptus by C. albidus (biopulping) was effective in reducing its lignin content and can be used for biopulping in the pulp and paper industry.

A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan.

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.

Herbarone, a rearranged heptaketide derivative from the sea hare associated fungus Torula herbarum.

Herbarone (1), a novel heptaketide with a tetrahydro-5,9-methanobenzo[8]annulen-10(5H)-one skeleton, together with the new ent-astropaquinones B (2) and C (3) and four known pyranonaphthoquinones (4-7), was isolated from the sea hare associated fungus Torula herbarum. The structures of the new compounds were elucidated by detailed spectroscopic analysis, and the absolute configurations were determined by solution TDDFT/ECD calculations. Absolute configurations of the known compounds were studied by ECD measurements and calculations. The isolation of heptaketide 1 suggests that an intramolecular aldol reaction takes place to form the tricyclic scaffold.

Cryptococcus laurentii biofilms: structure, development and antifungal drug resistance.

A great number of fungal infections are related to biofilm formation on inert or biological surfaces, which are recalcitrant to most treatments and cause human mortality. Cryptococcus laurentii has been diagnosed as the aetiological pathogen able to cause human infections mainly in immunosuppressed patients and the spectrum of clinical manifestations ranges from skin lesions to fungaemia. The effect of temperature, pH and surface preconditioning on C. laurentii biofilm formation was determined by 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay. Scanning electron microscopic (SEM) analysis of C. laurentii biofilms demonstrated surface topographies of profuse growth and dense colonization with extensive polymeric substances around the cells. In this study, we determined the activity of amphotericin B, itraconazole and fluconazole against C. laurentii free-living cells and biofilms. The activity of antifungals tested was greater against free-living cells, but sessile cells fell into the resistant range for these antifungal agents. Extracellular polymeric substances (EPS), comprising the matrix of C. laurentii biofilms, were isolated by ultrasonication. Fourier transform infrared spectroscopy (FT-IR) was performed with ethanol-precipitated and dried samples. Also, the multielement analysis of the EPS was performed by inductively coupled plasma optical emission spectroscopy (ICP-OES).

[Purification and physico-chemical properties of Cryptococcus albidus 1001 alpha-L-rhamnosidase].

A scheme of isolation and purification of the enzyme with alpha-L-rhamnosidase activity from culture liquid Cryptococcus albidus 1001 has been developed. It included fractionation by ammonium sulfate and chromatography on TSK-gels Toyopearl HW-60, Fractogel TSK DEAE-650-s and Sepharose 6B. The enzyme was purified 42 times with the yield of 0.7 %. The enzyme preparation did not contain any glycosidase (except of beta-D-glucosidase) and proteolytic activity. Molecular mass of the alpha-L-rhamnosidase preparation by the data of Sepharose 6B gel-filtration was 50 kDa. The enzyme preparation was stable during 48 hours at 20 degrees C, its pH and thermal optimum were 4.0-5.0 and 60 degrees C, correspondingly.

[Investigation of functional groups of Cryptococcus albidus alpha-L-rhamnosidase].

The effect of cations, anions and specific chemical reagents: 1-[3-(dimethylamino)propyl]-3-ethylcarbodimide methiodide, EDTA, o-phenantroline, dithiotreitol, L-cysteine, beta-mercaptoethanol, p-chlormercurybenzoate (p-ChMB), N-ethylmaleimide on the alpha-L-rhamnosidase activity of Cryptococcus albidus has been investigated. The essential role of Ag+ which inhibits the alpha-L-rhamnosidase activity by 72.5% was shown. Rhamnose at 1-5 mM protect the enzyme from the negative effect of Ag(+). It was expected that carboxyl group of C-terminal aminoacid and imidazole group of histidine would participate in the catalytic action of alpha-L-rhamnosidase on the basis of inhibition and kinetic analysis.

Analysis of Cryptococcal Extracellular Vesicles: Experimental Approaches for Studying Their Diversity Among Multiple Isolates, Kinetics of Production, Methods of Separation, and Detection in Cultures of Titan Cells.

Extracellular vesicles (EVs) produced by members of the Cryptococcus genus are associated with fundamental processes of fungal physiology and virulence. However, several questions about the properties of cryptococcal EVs remain unanswered, mostly because of technical limitations. We recently described a fast and efficient protocol of high-yield EV isolation from solid medium. In this study, we aimed at using the solid medium protocol to address some of the open questions about EVs, including the kinetics of EV production, the diversity of EVs produced by multiple isolates under different culture conditions, the separation of vesicles in a density gradient followed by the recovery of functional EVs, the direct detection of EVs in culture supernatants, and the production of vesicles in solid cultures of Titan cells. Our results indicate that the production of EVs is directly impacted by the culture medium and time of growth, resulting in variable detection of EVs per cell and a peak of EV detection at 24 h of growth. Nanoparticle tracking analysis (NTA) of EV samples revealed that multiple isolates produce vesicles with variable properties, including particles of diverging dimensions. EVs were produced in the solid medium in amounts that were separated on a centrifugation density gradient, resulting in the recovery of functional EVs containing the major cryptococcal capsular antigen. We also optimized the solid medium protocol for induction of the formation of Titan cells, and analyzed the production of EVs by NTA and transmission electron microscopy. This analysis confirmed that EVs were isolated from solid cultures of cryptococcal enlarged cells. With these approaches, we expect to implement simple methods that will facilitate the analysis of EVs produced by fungal cells. IMPORTANCE Fungal extracellular vesicles (EVs) are considered to be important players in the biology of fungal pathogens. However, the limitations in the methodological approaches to studying fungal EVs impair the expansion of knowledge in this field. In the present study, we used the Cryptococcus genus as a model for the study of EVs. We explored the simplification of protocols for EV analysis, which helped us to address some important, but still unanswered, questions about fungal EVs.

Treatment strategies for cryptococcal infection: challenges, advances and future outlook.

Cryptococcus spp., in particular Cryptococcus neoformans and Cryptococcus gattii, have an enormous impact on human health worldwide. The global burden of cryptococcal meningitis is almost a quarter of a million cases and 181,000 deaths annually, with mortality rates of 100% if infections remain untreated. Despite these alarming statistics, treatment options for cryptococcosis remain limited, with only three major classes of drugs approved for clinical use. Exacerbating the public health burden is the fact that the only new class of antifungal drugs developed in decades, the echinocandins, displays negligible antifungal activity against Cryptococcus spp., and the efficacy of the remaining therapeutics is hampered by host toxicity and pathogen resistance. Here, we describe the current arsenal of antifungal agents and the treatment strategies employed to manage cryptococcal disease. We further elaborate on the recent advances in our understanding of the intrinsic and adaptive resistance mechanisms that are utilized by Cryptococcus spp. to evade therapeutic treatments. Finally, we review potential therapeutic strategies, including combination therapy, the targeting of virulence traits, impairing stress response pathways and modulating host immunity, to effectively treat infections caused by Cryptococcus spp. Overall, understanding of the mechanisms that regulate anti-cryptococcal drug resistance, coupled with advances in genomics technologies and high-throughput screening methodologies, will catalyse innovation and accelerate antifungal drug discovery.

Torula yeast has greater digestibility of amino acids and phosphorus, but not energy, compared with a commercial source of fish meal fed to weanling pigs.

Three experiments were conducted to test the hypothesis that the standardized ileal digestibility (SID) of AA, concentrations of DE and ME, and the standardized total tract digestibility (STTD) of P in a source of torula yeast are not different from values obtained in Menhaden fish meal. In experiment 1, six weanling barrows (initial BW: 11.7 ± 0.4 kg) were prepared with a T-cannula in the distal ileum and allotted to a replicated 3 × 3 Latin square design with 3 diets and 3 periods. In each period, there were 5 d of adaptation and 2 d of collection. Two cornstarch-based diets using the torula yeast or fish meal as the sole source of AA and a N-free diet were formulated. The SID of CP and all AA was greater (P < 0.05) in torula yeast than in fish meal. In experiment 2, 24 weanling barrows (initial BW: 14.4 ± 1.1 kg) were individually housed in metabolism crates and allotted to a corn-based diet or 2 diets based on a mixture of corn and torula yeast or corn and fish meal. Feces and urine samples were collected for 4 d following a 5-d adaptation period. There were 8 replicate pigs per diet and fecal and urine materials were collected. Results of this experiment indicated that there were no differences in the concentration of DE and ME (DM basis) between torula yeast and fish meal. In experiment 3, a total of 32 weanling barrows (initial BW: 11.9 ± 1.1 kg) were allotted to 4 diets and 8 replicate pigs per diet. Pigs were placed in individual metabolism crates. The torula yeast or fish meal were used in 2 diets containing either 0 or 500 units of microbial phytase. Feces samples were collected as described for experiment 2. The STTD of P in torula yeast was greater (P < 0.05) than in fish meal, but regardless of ingredient, there was no effect of the inclusion of phytase in the diets. In conclusion, the SID of AA and the STTD of P in torula yeast is greater than in fish meal, but values for the concentration of DE and ME in torula yeast are not different from those in fish meal. Therefore, the torula yeast that was used in the present experiments may be included at the expense of fish meal in diets fed to weanling pigs if the concentration of standardized ileal digestible AA is considered in the formulation.

A highly thermosensitive and permeable mutant of the marine yeast Cryptococcus aureus G7a potentially useful for single-cell protein production and its nutritive components.

The highly thermosensitive and permeable mutants are the mutants from which intracellular contents including proteins can be released when they are incubated both in the low osmolarity water and at the nonpermissive temperature (usually 37 degrees C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named Z114 was obtained from the marine yeast Cryptococcus aureus G7a. Of the total protein, 65.3% was released from the mutant cells suspended in distilled water after they were treated at 37 degrees C overnight. However, only 12.3% of the total protein was released from the mutant cells suspended in 1.0 M sorbitol solution after they were treated at 37 degrees C overnight. We found that intracellular density of the mutant treated at 37 degrees C was greatly decreased, and cell volume of the mutant treated at 37 degrees C was increased due to the increased protein release. However, no significant changes in the intracellular density and cell volume of the mutant were observed when its cells suspended in 1.0 M sorbitol solution were treated at 37 degrees C. It was found that no big changes in cell growth, protein content, vitamin C content, nucleic acid content, fatty acids, and amino acid compositions of both the mutant and its wild type were detected. Therefore, the highly thermosensitive and permeable mutant still can be a good candidate as single-cell protein. This means that the method used in this study is a simple and efficient way to release protein from the highly thermosensitive and permeable yeast mutant cells with high protein content.

Growth and pigment production on D-tryptophan medium by Cryptococcus gattii, Cryptococcus neoformans, and Candida albicans.

Given the increasing prevalence of cryptococcosis caused by Cryptococcus gattii (serotypes B and C) strains, there is a need for rapid and reliable tests that discriminate C. gattii from Cryptococcus neoformans (serotypes A, D, and AD). Seventy-two C. neoformans strains, sixty-seven C. gattii strains, and five Candida albicans strains were analyzed for their ability to grow and produce pigment on minimal D-tryptophan D-proline (m-DTDP) medium, on yeast carbon base D-tryptophan D-proline (YCB-DTDP) medium, and on fructose D-tryptophan glycine (m-FDTG) medium. Of the C. gattii and C. neoformans isolates, 94% and 0% grew on m-DTDP agar, respectively, and 98% and 0% grew in YCB-DTDP medium, respectively. C. gattii produced large amounts of brown intracellular pigment(s) on m-DTDP agar and smaller amounts of yellow-brown (amber) extracellular pigment(s). C. albicans grew on both media and produced a pink photoactivated pigment on m-DTDP agar. C. gattii produced large amounts of brown intracellular pigments on the differential medium m-FDTG, whereas C. neoformans produced smaller amounts of the brown pigments and C. albicans produced a pink pigment. The pigments produced by C. gattii from D-tryptophan were distinct and were not related to melanin formation from 3,4-dihydroxyphenylalanine. Thin-layer chromatography of the methanol-extracted C. gattii cells detected four different pigments, including brown (two types), yellow, and pink-purple compounds. We conclude that tryptophan-derived pigments are not melanins and that growth on m-DTDP or YCB-DTDP agar can be used to rapidly differentiate C. gattii from C. neoformans.

Identification and determination of selenohomolanthionine - The major selenium compound in Torula yeast.

Torula yeast (Candida utilis) was found to metabolize selenium in a totally different way to Brewer's yeast (S. cerevisiae) leading to the biosynthesis of selenohomolanthionine (SeHLan), a major selenium compound accounting for 60-80% of the total selenium. The identity of SeHLan was confirmed by retention time matching in hydrophilic ion interaction chromatography (HILIC) with inductively coupled plasma mass spectrometric detection (ICP MS) using a custom synthesized standard molecule and by HILIC - Orbitrap MS and MS-MS fragmentation. Selenohomolanthionine escapes the current assays for the organic character of Se-rich yeast based on the protein-bound selenomethionine determination. A HILIC - ICP MS method was developed for the quantitative determination of selenohomolanthionine in yeast supplements with a detection limit of 146ng/g.

An improvised medium for axenic cultivation of Acanthamoeba spp.

Acanthamoebae can be easily grown in bacterised cultures, but their growth in axenic media is tedious and many times unsuccessful. We thus experimented with some additives in the conventional axenic medium for growth of various isolates of Acanthamoeba. Addition of Torula yeast RNA was found to significantly enhance the growth of Acanthamoebae in the axenic culture medium.

Effects of Cryptococcus humicola killer toxin upon Cryptococcus terreus envelope: combined fluorometric and microscopic studies.

Killer toxin (microcin) produced by Cryptococcus humicola 9-6 induced interaction of the fluorogenic dyes, ethidium bromide, propidium iodide, and hemimagnesium 8-anilino-1-naphtalenesulfonate, with the sensitive strain of Cryptococcus terreus VKM Y-2253. The toxin also made the cells susceptible to cetyltrimethylammonium bromide and leaky for K+. When excited at 360 nm, cell-bound ethidium (propidium) fluorescence was enhanced by 8-anilino-1-naphtalensulfonate, and cell-bound 8-anilino-1-naphtalensulfonate fluorescence was quenched by ethidium (propidium), indicating energy transfer from 8-anilino-1-naphtalensulfonate to ethidium (propidium). These results suggest that at least a portion of the probe molecules had the same binding site, possibly the cytoplasmic membrane. The parameters of kinetics of microcin action were evaluated fluorometrically. They were found to be identical for all probes and depended on microcin concentration. The fluorescence increment of ethidium and 8-anilino-1-naphtalensulfonate upon binding to microcin-treated cells correlated with the fraction of stainable cells and viability.

The mycocidal, membrane-active complex of Cryptococcus humicola is a new type of cellobiose lipid with detergent features.

The chemical composition of the mycocidal complex (formerly known as microcin) secreted by Cryptococcus humicola was investigated by chemical, mass spectrometric and nuclear magnetic resonance methods. The results indicate that the mycocidal complex is composed of glycolipids with a highly acetylated (up to five acetyl groups) cellobiose backbone [beta-D-Glcp-(1'-->4)-beta-D-Glcp] linked to the omega-hydroxyl group of alpha,omega-dihydroxy palmitate [16:0-alpha,omega-di-OH] with an unsubstituted carboxyl group. The acyl chain forming aglycon can be replaced by [18:0-(alpha,omega-di-OH)], [18:0-(alpha,omega-1,omega-tri-OH)], and [18:0-(alpha,omega-2,omega-tri-OH)]. The complex has a comparatively high surface activity; 0.5 mg/ml of it reduced the surface tension of 0.1 M NaHCO(3) from 71 mN/m to 37 mN/m and interfacial tension against n-hexadecane from 39 mN/m to 10 mN/m. The critical micelle concentration of the complex at pH 4.0, determined by the fluorometric method with N-phenyl-1-naphthylamine as fluorescent probe and by the De Nouy ring method, was 2 x 10(-5) M (taking the average molecular mass of the complex to be 750); it did not depend on the presence of 100 mM KCl and was an order of magnitude higher at pH 7.0. By fluorescence resonance energy transfer spectroscopy with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine as energy donor and N-(rhodamine B sulfonyl)-phosphatidylethanolamine as energy acceptor the complex was shown to intercalate into the liposomal lipid matrix. Primary lesions caused by the complex in planar lipid bilayers were revealed as short-living current fluctuations of a broad spectrum of amplitudes. The mycocidal effect of the complex is suggested to be associated with its detergent-like properties.